Pronase purification

ABSTRACT

The antigenic materials in 6-aminopenicillanic acid and penicillins and derivatives thereof obtained by fermentation are degraded by bringing an aqueous solution of the 6aminopenicillanic acid or penicillin or derivatives thereof into contact with water-insoluble derivative of a proteolytic enzyme. The proteolytic enzyme is preferably pronase and the waterinsoluble derivative is obtained from the enzyme and bromoacetylcellulose or from the enzyme and sephadex or carboxymethylcellulose. The resulting product is unlikely to cause anaphylactic shock.

I United States Patent 1191 1111 3,770,584

Shaltiel et al. 1 Nov. 6, 1973 PRONASE PURIFICATION OTHER PUBLICATIONS [75] Inventors: Shmuel Shaltrel; Michael Sela, both Knudsen, The Lancet June 1967, pages 344 p of Israel Silman et 61., Annual Review Of Biochemistry 1966, [73] Assignee: Beecham Group Limited, Brentford, VOL 35 P p g Middlesex, En land Weliky et al., lmmunochemistr Dec. 1965, a es g 319 320 y p g [22] plied: May 1972 Kay, Process Biochemistry, Aug. 1968 pages 36-39. [21] Appl. No.: 252,051

Primar Examiner-Lionel M. Sha iro Related US. Application Data y p [63] Continuation of Sfil'. NO. 802,688, Feb. 26, 1969, Ammey jacobs Jacobs b 1 1. a 57 ABSTRACT [30] Foreign Application Priority Data The antigenic materials in 6-aminopenicillanic acid and Feb. 29, 1968 Great Britain 9,778/68 peniti"ins and derivatives thereof Obtained y fermentation are degraded by bringing an aqueous solution of [52] US Cl. 195/2, 195/36 P, l95/DIG, 11 the 6-aminopenicillanic acid or penicillin or derivatives 51 Int. Cl Cl2d 9/12 thereof into Contact with water-insoluble derivative of [58] Field 61 Search 195/63, 36 P, DIG. 11, apmteolytic enzyme- The Pmteolyttc enzyme is P 195/2, 63 ably pronase and the water-insoluble derivative is obtained from the enzyme and brornoacetyl-cellulose or [56] References Cited from the enzyme and sephadex or carboxymethyl- UNITED STATES PATENTS cellulose. The resulting product is unlikely to cause anaphylactic shock. 3,556,945 1/1971 Messmg 195/63 3,446,705 5/1969 Heuser et al. 195/36 P 3 Claims, N0 Drawmgs PRONASE PURIFICATION This is a continuation, Ser. No. 802,688 filed Feb. 26, 1969, now abandoned.

The present invention relates to a method of reducing the antigencity associated with 6-aminopenicillanic acid and penicillins and derivatives thereof obtained by fermentation processes.

6-Aminopenicillanic acid and penicillins and derivatives thereof. obtained be fermentation processes as hereinafter defined, and as isolated by conventional methods contain strongly antigenic impurities. These impurities cause known disadvantages when the penicillins (which may have been prepared from the 6- aminopenicillanic acid) are used as therapeutic agents, for example they may produce anaphylactic shock in patients. It is therefore an object of the present invention to provide a process for degrading these antigenic impurities so that the immunogenicity of the product and its reactivity with specific antibodies are very much reduced.

Accordingly the present invention provides a process for degrading the antigenic materials in 6- aminopcnicillanic acid and penicillins and derivatives thereof obtained by a fermentation method as hereinafter defined, which process comprises contacting an aqueous solution containing said 6 aminopenicillanic acid or penicillin or derivative thereof with at least one water-insoluble derivative of at least one proteolytic enzyme.

It is to be understood that the degradation process may be applied to (l) penicillins produced by direct fermentation in presence of a side-chain precursor, (2) 6-aminopenicillanic acid prepared by direct fermentation in a synthetic nutrient medium, (3) 6- aminopcnicillanic acid prepared by enzymatic hydrolysis of a penicillin, (4) 6-aminopenicillanic acid or an ester thereof prepared by chemical removal of the side chain from a penicillin, or 5) penicillins or derivatives thereof prepared from 6-aminopenicillanic acid not previously treated by the degradation process; the penicillins could be prepared by straight-forward acylation of the 6-aminopenicillanic acid and purified directly or converted to closely related derivatives, for example the reaction product of a-aminobenzylpenicillin with acetone, which derivative is subjected to the degradation process.

The water-insoluble enzyme derivatives are prepared by, known methods, for example by the methods described in British Patent Specifications Nos. 916,931

7 and 1,062,596. Preferably the proteolytic enzyme used and has a broad specificity. The enzyme may be reacted for example with bromoacetylcellulose or coupled with CM-Sephadex (Sephadex is a registered trade mark) in presence of a suitable amide-forming reagent, such as a carbodiimide. The water-insoluble derivatives retain the activity of the parent enzyme, but in use remain in the water-insoluble form, and so may be used in continuous processes in columns and other equipment. More than one enzyme may be present in the water-soluble form to increase the range of activity, with consequent greater degradation of the proteinaceous antigens in the starting material.

With water-insoluble pronase, the process is preferably effected at pH 7-8 for optimum results, and the diperiments showed that the pronase lacked specificitygestion of proteinaceous materials occurs rapidly, probably in l to 3 hours.

The efficacy of pronase to degrade the proteinaceous antigenic impurities found in 6-aminopenicillanic acid was shown by tests made with an isolated antigenic fraction, that was treated with pronase. The tests involved electrophoresis and chromatography of the treated fraction and showed that it behaved like an amino acid mixture. The treated fraction on dialysis left only a retentate of pronase, whereas the initial fraction was not dialysable in a control test. By the PCA test it was established that no antibodies had been produced in animals injected with the treated fraction. The efficacy of the pronase was not affected when the antigenic fraction was mixed with a large excess of 6- aminopcnicillanic acid, i.e., to correspond with the situation in commercial 6-aminopenicillanic acid.

Similar results on the efficacy of water-insoluble derivatives of pronase were found in tests with batches of 6-aminopenicillanic acid and such penicillins as benzylpenicillin and aaminobenzylpenicillin.

The following Examples illustrate the invention:

EXAMPLE 1 The antigenic impurity fractions previously isolated from commercial 6-aminopenicillanic acid by using a molecular sieve were tested as follows:

One 1.5 mg. of the fraction was dissolved in 5.7-N hydrochloric acid and hydrolysed at 1C for 16 hours. The product was examined with an amino acid analyser.

Another 1.5 mg. of the fraction was dissolved in 0.5 ml. of 0.05 M ammonium bicarbonate buffer at pH 7.9 and 107 of a solution of 1.5 mg. pronase/ml. in same buffer was added and the mixture was incubated at 37C for 16 hours, then evaporated to dryness and the product was also examined with an amino acid analyser.

For both products the presence of a large number of amino acids was determined and some small peptides, probably dipeptides, appeared to. be formed. The ex- EXAMPLE 2 2 mg. of the antigenic impurity fraction (previously isolated from commercial 6-aminopenicillanic acid by using a molecular sieve) in 1 ml. of 0.05 M ammonium bicarbonate buffer atpl-l 8.3 was mixed with 0.02 mg. commercially'available water-soluble pronase and was incubated at 37C for 22 hours.

The digest had a considerably reduced ability to provoke the formation of antibodies as indicated by PCA tests in the guinea pig. In control tests (1) the untreated antigenic impurity fraction in the buffer and freshly prepared before injection and (2) 2 mg. of the antigenic impurity fraction treated in the same way as described above but without addition of pronase, were used.

The results are shown in the following Table I.

TABLE I No. of positive Time Group animals per total Diameter (days) No. of animals of spot (mm) 10 A /4 0 B a 0/4 0 C 0/10 0 18 A 2/4 B 1/3 10 C 0/9 0 24 A 3/4 25 B 2/3 25 C 0/9 0 54 A 3/3 30 B 3/3 30 C 1/8 23 90 A 3/3 26 B 3/3 28 C 2/7 18 134 A 3/3 27 B 3/3 28 C 2/7 20 Awntreated antigenic impurity fraction B=fraction incubated without pronase C=fraction incubated with pronase The experiment involved three groups of guinea pigs, groups A and 8 each involving four guinea pigs and group C involving 10 guinea pigs.

Animals from group A were immunised with 0.25 ml. of the incubated mixture from the antigenic impurity fraction and pronase, mixed with 0.25 ml. of Freunds complete adjuvant. Animals from groups B and C were immunised similarly with solutions prepared for the control tests as described above.

Bleedings were carried out at the indicated intervals and the sera obtained were frozen until usage. Passive cutaneous anaphylaxis (PCA) tests were carried out on albino guinea pigs (weighing 250-300g.) Amounts of 0.1 ml. antiserum were injected intradermally and 18 hours later the animals were challenged with 1 ml. of a freshly prepared solution of the untreated antigenic impurity fraction (1 mg./ml. in saline), mixed with 1 ml. of a 1 percent solution of Evans Blue. The diameters of the spots were recorded after 10 minutes.

EXAMPLE 3 The effect of pronase on the antigenicity of an antigenic impurity fraction (previously isolated from commercial 6-aminopenicillanic acid by using a molecular sieve material) was determined in skin tests, and the results are shown in Table II.

Average diameter of reaction Amount of antigen Eight guinea pigs were immunised with the untreated impurity fraction as described in Example 2. After 20 days they reached a uniform antibody level in the serum determined by PCA tests. At that time each animal was skin tested with 0.05-50y of the impurityfraction in 0.1 ml. saline or an equivalent amount of the fraction previously digested with pronase (dissolved in 0.05 M-ammonium bicarbonate, treated with pronase and incubated for 22 hours at 37C, as described in Example 2). Reaction spots were measured 24 hours after challenge.

EXAMPLE 4 Similar experiments to those described in Example 2 were made with mixtures of (A) 6-aminopenicillanic acid and 1 percent of the antigenic impurity fraction and (B) 6-aminopenicillanic acid, 1 percent antigenic impurity fraction and pronase (2 percent based on the protein content of the impurity fraction), and the results are shown in Table 111.

immunogenicity was assessed by PCA tests as described in Example 2.

EXAMPLE 5 The effect of pronase on the titration of antisera against an antigenic impurity fraction from commercial -aminopenicillanic acid was determined and the results are shown in Table IV.

TABLE IV Average diameter of spot (mrn) Dilution of serum Untreated Pronase treated 1:5 26 21 1:10 25 17 1:20 21 14 1:50 19 12 1:100 17 10 end int end point 1:2000

ControlzNor-rnal Guinea Pig serum 0 0 diluted 1:5

A pool of positive antisera against the impurity fraction was diluted with saline as shown in the Table. A volume of 0.1 ml. of the diluted solution was injected intradermally to two groups each of four guinea pigs. One of the groups was challenged after 18 hours with 1 ml. of solution containing 0.1 mg. of the impurity fraction and 1 ml. Evans blue in saline. The other group was challenged with an equivalent amount of impurity fraction treated with water-insoluble pronase (obtained by reaction of pronase with bromoacetylcellulose).

EXAMPLE 6 Samples of commercial benzylpenicillin and ampicillin were tested for reduction of immunogenicity after treatment with water-insoluble pronase (from bromoacetylcellulose and pronase), and the results are shown in Table V.

TABLE V Time No. of positive Compound (days) Group animals per total Average number of survivdiameter ing rabbits of spot immunized (mm) Penicillin 14 A 2/5 20 G C /5 0 Do. 21 A /5 24 C 1/5 25 Do. 28 A 4/5 26 C [/5 23 Ampicillin 14 A 2/2 33 C 0/3 0 Do. 21 A 2/2 29 C 0/3 0 Do. 28 A 2/2 30 C 0/3 0 Groups of 5 rabbits were immunized with 50 mg. of the test penicillin. Groups A received untreated samples of the penicillins while groups C received the penicillins after prior incubation with an equal weight of insoluble pronase (at 37C for 22 hours in buffer at pH 8.3). Injections were given subcutaneously in complete Freunds adjuvant (1:1). Booster injections of 50 mg. antigen in complete Freunds adjuvant were given on the 8th and 15th day. The rabbits were bled on days 1, 7, 14, 21 and 28 and the sera were stored at -C. PCA tests were carried out on the antisera using penicilloyl BGG as antigen.

EXAMPLE 7 300 mg. pronase in citrate phosphate buffer at pH 5.1 was added to 10 g. bromoacetyl-cellulose (wet weight). The suspension was stirred gently for 30 hours at room temperature (22C) and drop of antifoam agent was added. The suspension was then centrifuged at 10,000 G for 10 minutes and the precipitate was resuspended in 30 ml. 0.1 M bicarbonate buffer at pH 8.9. The suspension was left at 4C for 24 hours and then centrifuged at 10,000 G for 10 minutes. The supernatants were kept for measurement of optical density. The precipitate was'resuspended in 0.05 MZ-aminoethanol in 0.1 M bicarbonate buffer at pH 8.9. The preparation was finally centrifuged again and washed in 0.15 M sodium chloride until the supernatant had no optical density at 280 mu. 1 Y

The catalytic activity of the bound pronase was determined by using as substrate tyrosyltyrosine, tryptophyltyrosine or leucyl glycine. The activity against these substrates was determined chromatographically as a function of time, with soluble pronase as control.

EXAMPLE 8 1.5 mg. of an impurity fraction from a sample of commercial 6-aminopenicillanic acid was dissolved in 0.5 ml. 0.05 M ammonium bicarbonate bufier at pH 7.9 and added to 10 7 (0.01 ml.) of a solution containing 1.5 mg. pronase/ml. (as water-insoluble material) in the same buffer. The mixture was incubated at 37C for 16 hours with gentle mixing and then evaporated to dryness.

in such a test it was found that the water-insoluble pronase material could be used repeatedly without appararent loss of activity. For example, material obtained from bromoacetylcellulose was used five times during 10 months. Each time after use the material was washed in buffer then in saline and stored at 4C in presence of calciumions to stabilise the enzyme.

EXAMPLE 9 A 2g. sample of commercial 6-aminopenicillanic acid was dissolved in water with addition of 3N-sodium hydroxide to pH 8.6, then diluted to 20 ml. The solution was divided into two equal parts, both of which were incubated at 37C for 20 hours, except that one part contained 500 mg. of water-insoluble pronase material (from pronase and bromoacetylcellulose). The solution containing the pronase was centrifuged, and the two solutions were adjusted to pH 4.3 with 3N-hydrochloric acid and kept at 0C for 24 hours. Solid 6- aminopenicillanic acid was recovered from each solution and used to immunise rabbits according to the following schedule and the antibody response was measured:

Day 1 50 mg. of the -aminopenicillanic acid in Freunds complete adjuvant (2 mg./ml. M. butyricum) 6 mg. of the 6-aminopenicillanic acid in aq. solution (at 6 X 0.1 ml. of a 10 mg./ml. solution), all subcutaneously.

Day 7 40 mg. of the 6-aminopenicillanic acid subcutaneously in Freunds complete adjuvant.

The results are shown in Table VI.

TABLE VI Haemagglutination titre.

Reciprocal against benzyl- Material Rabbit penicilloylated rabbit erythrocytes Day 0 7 14 21 28 Control 1 0 2048 16400 512 128 6-aminopenicillanic 2 0 256 512 200 256 acid Pronase- 4 0 6 128 32 64 treated 6-arninopen- 5 0 4 4 8 8 icillanic acid 6 0 0 0 4 128 The results showed that there was a significant reduction in immunogenicity of the commercial 6- aminopenicillanic acid (which was a highly immunogenic batch) after the treatment with insoluble pronase.

We claim:

1. A process for reducing the antigenicity of the antigenic materials in 6-aminopenicillanic acid and penicillins and derivatives thereof obtained by a fermentation method prior to administration thereof, which process comprises chemically degrading the antigens but not the -amino-penicillanic acid and penicillins and derivatives thereof by contacting an aqueous solution containing said 6-aminopenicillanic acid or penicillin or derivative thereof with at least one water-insoluble derivative of pronase which retains the activity of the pronase and is obtained by coupling the pronase and CM- sephadex or carboxymethylcellulose.

2. A process as claimed in claim 1, wherein the degradation is effected at pH 78.

3. The process according to claim 1, wherein the water-insoluble derivative is obtained by reacting the pronase and bromoacetylcellulose. 

2. A process as claimed in claim 1, wherein the degradation is effected at pH 7-8.
 3. The process according to claim 1, wherein the water-insoluble derivative is obtained by reacting the pronase and bromoacetylcellulose. 